School of Optometry and Ophthalmology and Eye Hospital, Institute of Molecular Medicine, Wenzhou Medical UniversityWenzhou, China; Department of Neurology, School of Medicine, Boston UniversityBoston, MA, USA.

Abstract

Striatal adenosine A2A receptors (A2ARs) modulate striatal synaptic plasticity and instrumental learning, possibly by functional interaction with the dopamine D2 receptors (D2Rs) and metabotropic glutamate receptors 5 (mGluR5) through receptor-receptor heterodimers, but in vivo evidence for these interactions is lacking. Using in situ proximity ligation assay (PLA), we studied the subregional distribution of the A2AR-D2R and A2AR-mGluR5 heterodimer complexes in the striatum and their adaptive changes over the random interval and random ratio training of instrumental learning. After confirming the specificity of the PLA detection of the A2AR-D2R heterodimers with the A2AR knockout and D2R knockout mice, we detected a heterogeneous distribution of the A2AR-D2R heterodimer complexes in the striatum, being more abundant in the dorsolateral than the dorsomedial striatum. Importantly, habit formation after the random interval training was associated with the increased formation of the A2AR-D2R heterodimer complexes, with prominant increase in the dorsomedial striatum. Conversely, goal-directed behavior after the random ratio schedule was not associated with the adaptive change in the A2AR-D2R heterodimer complexes. In contrast to the A2AR-D2R heterodimers, the A2AR-mGluR5 heterodimers showed neither subregional variation in the striatum nor adaptive changes over either the random ratio (RR) or random interval (RI) training of instrumental learning. These findings suggest that development of habit formation is associated with increased formation of the A2AR-D2R heterodimer protein complexes which may lead to reduced dependence on D2R signaling in the striatum.

Detection and specificity of the A2AR-D2R and A2AR-mGluR5 heterodimers in the striatum by PLA. (A) Representative immunofluorescent photomicrographs show A2ARs and D2R were highly expressed in the striatum of WT mice, but were absent in A2AR or D2R KO mice. (B) Physical association of A2AR-D2R heterodimers was detected by PLA in sections of the striatum from WT mice, but not from A2AR KO mice and D2R KO mice. The enlarged confocal image (Left) identified the PLA signals (red puncta) and positive cells (white arrows). (C) Quantification of the A2AR-D2R heterodimers by PLA in the striatum of WT (n = 6), A2AR KO (n = 3), and D2R KO (n = 3) mice. (D) PLA signals of A2AR-mGluR5 heterodimers were detected in the striatum of WT mice (middle), with amplified image (left), but were absent in A2AR KO mice (right). (E) Quantification of A2AR-mGluR5 heterodimers by PLA for WT (n = 6) and A2AR KO (n = 3) mice. Data are presented as the mean ± SEM.

Heterogeneous distribution of the A2AR-D2R heterodimers (but not the A2AR-mGluR5 heterodimers) between DMS and DLS. (A) Representative images show: three sections from one brain which was from anterior, middle, and posterior parts of the striatum, respectively. (B) Left: representative images show that the A2AR-D2R heterodimers (as indicated by white arrows) were more abundant in the DLS (lower panels) than DMS (upper panels). Right: quantification of A2AR-D2R heterodimers confirms that the A2AR-D2R heterodimers was more abundant in DLS than DMS in the striatum (**p = 0.003, paired t-test). (C) Left: representative images show the A2AR-mGluR5 heterodimers (as indicated by white arrows) were indistinctive in both DMS and DLS. Right: quantification of the A2AR-mGluR5 heterodimers shows that the A2AR-mGluR5 heterodimers displayed no subregional variation between DMS and DLS (p = 0.612, paired t-test). Data are presented as the mean ± SEM, n = 6/group.